The integrins mediate adhesive interactions between cells and between cells and the extracellular matrix. Platelet adhesion to subendothelium and other platelets is in large part mediated by integrins. The alpha2beta1 integrin serves as a receptor for collagen on platelets and fibroblasts and as a receptor for collagen and laminin on endothelial and epithelial cells. Although recent studies have elucidated the structure and function of integrin receptors and their ligands on platelets, the regulation of integrin gene expression, important in megakaryocytic differentiation, remains largely unexplored. We have demonstrated that increased surface expression of functional alpha2beta1 integrin which accompanies the megakaryocytic differentiation of K562 cells is a consequence of increased steady-state levels of alpha2 mRNA, primarily due to increased transcription of the alpha2 integrin gene. The time course of alpha2 induction was slow (measured in days) and obliterated by cycloheximide, which suggests a requirement for new protein synthesis of transcriptional regulators prior to the synthesis of alpha2 mRNA. This system is well-suited to explore the regulation of integrin gene expression during megakaryocytic differentiation. To pursue the regulation of the alpha2 integrin during megakaryocytic differentiation, we have recently identified the 5' flanking region of the alpha2 gene and characterized the transcription start site. The experiments described in this proposal are designed to elucidate the molecular mechanisms of phorbol ester-induced and megakaryocyte-specific regulation of alpha2 integrin gene expression. The promoter and enhancer activity of the 5' flanking region of the alpha2 gene will be established by the ability to direct transcription of the bacterial chloramphenicol acetyl transferase gene in our model system, as well as in the megakaryocytic cell lines DAMI and HEL. Deletional and site-directed mutagenesis analyses will be used to define the regulatory elements. Mobility shift assays and DNA footprinting analysis will reveal the binding sites of transcriptional regulators conferring megakaryocyte-specific gene expression. The role of one family of transcriptional regulators, the Hox B genes, in regulating expression of the alpha2 integrin gene will be determined. The proposed experiments will lead to an initial understanding of the regulatory elements and proteins, giving rise to megakaryocyte-specific expression of the alpha2 integrin subunit.